Assay for cardiac hypertrophy

Abstract

An assay to test for hypertrophic activity in myocytes is described where wells are precoated with D-MEM/F-12 and fetal calf serum, plated with myocytes, cultured, and any change in size of the cells is determined. The growth medium may contain insulin, transferrin and aprotinin.

What is claimed is: 
     
       1. A method for assaying a test sample for hypertrophic activity in myocytes, the method comprising: (a) precoating a microwell with a precoating medium, wherein the precoating medium comprises D-MEM/F-12 further comprising 4% fetal calf serum;   (b) removing the precoating medium from the microwell;   (c) plating the microwell with a suspension of myocyte cells at a cell density of about 7.5×10 4  cells per mL in a serum free growth medium comprising D-MEM/F-12 medium further comprising insulin, transferrin, and a protease inhibitor;   (d) culturing the myocyte cells in the serum free growth medium, wherein the cells survive but do not undergo cell density-induced hypertrophy in the absence of the test sample;   (e) adding the test sample to the cultured myocyte cells of step (d);   (f) culturing the myocyte cells with the test sample in the serum free growth medium; and   (g) measuring for hypertrophic activity of the test sample by monitoring a physiological change in the cells wherein the chance is measured relative to control cells and the chance is selected from the group consisting of a change in the size of the myocyte cells and release of atrial natriuretic factor or atrial natriuretic protein from the myocyte cells.   
     
     
       2. The method of claim 1, wherein the plating in step (c) comprises plating a 96-well plate. 
     
     
       3. The method of claim 1, wherein the volume of the test sample is 100 μL. 
     
     
       4. The method of claim 1, wherein the test sample is selected from the group consisting of a cell supernatant, a cell lysate, a cell culture conditioned medium, or an isolated cell fraction. 
     
     
       5. The method of claim 1, further comprising adding cardiotrophin-1 (CT-1) or a CT-agonist to the microwell when the test sample is suspected of containing an antagonist. 
     
     
       6. The method of claim 5, wherein CT-1 is added to the microwell when the test sample is suspected of containing an antagonist. 
     
     
       7. The method of claim 1, wherein the measuring of step (g) comprises crystal violet staining prior to measuring of cell size. 
     
     
       8. The method of claim 1, wherein the protease inhibitor is aprotinin. 
     
     
       9. The method of claim 8, wherein the medium comprises 100 μL transferrin (10 mg/mL), 20 μL insulin (5 mg/mL), and 50 μL aprotinin (2 mg/mL) per 100 mL D-MEM/F-12. 
     
     
       10. The method of claim 1, wherein the medium is supplemented with a growth factor that promotes cell viability. 
     
     
       11. The method of claim 1, wherein the myocyte cells are cultured for at least 24 hours prior to adding test sample. 
     
     
       12. The method of claim 1, wherein the myocyte cells are cultured with test sample for 24 to 72 hours. 
     
     
       13. The method of claim 1, wherein precoating comprises incubating the microwell with the precoating call culture medium for eight hours at 37° C. 
     
     
       14. The method of claim 1, further comprising: (a) incubating the microwell in step (a) with the precoating medium for eight hours at 37° C.;   (b) culturing the myocyte cells in step (d) for at least 24 hours; and   (c) culturing the myocyte cells in step (f) for 48 hours.